Field of Invention
The present invention relates to methods and materials for biosynthesis of mogrol precursors, mogrol, and/or mogrosides. More particularly, the present invention relates to methods of using of cucurbitadienol synthase, cytochrome P450, cytochrome P450 reductase, and/or epoxide hydrolase enzymes to produce mogrol precursors and/or mogrol. The present invention also relates to methods of using of uridine-5′-diphospho (UDP) dependent glucosyltransferase (UGT) enzymes to glycosylate mogrol and produce various mogrosides.
Description of Related Art
Mogrosides are a family of triterpene glycosides isolated from fruit of Siraitia grosvenorii (S. grosvenorii, Swingle), also known as Momordica grosvenori. Fruit extracts are commercially used as natural sweeteners. Four major compounds, mogroside V, mogroside IV, siamenoside I, and 11-oxomogroside V (see FIG. 1) have been identified from S. grosvenorii as being responsible for the fruit's sweetness. Mogroside V is the most abundant of these four compounds, at approximately 0.57% (w/w) of the dry fruit, followed by mogroside IV and siamenoside I, each of which contains four glucose moieties. 11-oxomogroside V has a ketone group instead of a hydroxyl at C11. See, e.g., Takemoto et al., 1983, Yakugaku Zasshi 103: 1151-4; 1155-66; 1167-73; Kasai et al., 1989, Agric. Biol. Chem. 53:3347-9; Matsumoto Chem. Pharm. Bull., 1990, 38:2030-2; and Prakash et al., 2011, J. Carbohydrate Chem. 30:16-26.
All mogrosides share the same mogrol triterpene core. The aglycone mogrol is glycosylated with different numbers of glucose moieties to form various mogroside compounds. Mogrosides can be synthesized in the following manner: synthesis of cucurbitadienol from the common triterpene precursor oxidosqualene, oxidation of cucurbitadienol to produce mogrol, and glycosylation of mogrol to produce various mogrosides. See, Tang et al., BMC Genomics 12: 343 (2011). Tang et al., 2011, BMC Genomics 12:343 describes seven cytochrome P450s and five UGTs as potential candidates involved in mogroside biosynthesis. However, Tang et al. does not specifically identify any cytochrome P450s or UGTs involved in mogroside biosynthesis. Thus, there remains the need to identify cytochrome P450s and UGTs capable of acting on any S. grosvenorii metabolites. Additionally, although mogrosides can be extracted from S. grosvenorii, there remains a need for improved production of mogrosides in recombinant hosts for commercial uses.